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Method for preserving cells, tissues or organs in hypothermia

a technology for preserving cells and tissues, applied in the field of preserving cells, tissues or organs in hypothermia, can solve the problems of not being able to meet the needs of all cell types, unable to meet the needs of thawing, and not being able to achieve the survival and maintenance of cell functional capacities, so as to improve the survival and proliferative capacity of said cells

Active Publication Date: 2020-05-26
ESTAB FR DU SANG +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The patent text describes a method of preserving cells by first cooling them in a moderate way and then exposing them to a hypoxic and / or hypercapnic atmosphere before putting them in a severe cooling process. This method has been shown to improve the survival and ability of cells to grow when they are preserved. This is particularly useful for stem cells.

Problems solved by technology

Cryopreservation, however, has notable disadvantages, particularly potential agglutination during thawing or the risk of anaphylactic reaction triggered by the presence of cryoprotectants.
Moreover, this method is not applicable to all cell types.
Although notable, these improvements remain inadequate to ensure the survival and the maintenance of the functional capacities of cells, and in particular of progenitor cells, for long-term preservation at 4° C.

Method used

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  • Method for preserving cells, tissues or organs in hypothermia
  • Method for preserving cells, tissues or organs in hypothermia
  • Method for preserving cells, tissues or organs in hypothermia

Examples

Experimental program
Comparison scheme
Effect test

example 1

Placental blood CD34+ cells

Materials and Methods

[0081]After being isolated by an immunomagnetic system, CD34+ cells were incubated overnight in culture at 37° C. in HP01 medium (Macopharma) in the presence of stem cell factor (SCF; 100 ng / mL), thrombopoietin (20 ng / mL) and IL-3 (1 ng / mL) at a concentration of about 105 cells per mL. This medium ensures the survival of CD34+ cells without stimulating their proliferation. This culture was divided into 8 gas-impermeable flasks (2 per atmospheric condition: i) air (21% O2, 0.01% CO2); ii) hypoxia / hypercapnia (HH) (9% CO2, 5% O2); iii) hypoxia (5% O2, 0.001% CO2); iv) hypercapnia (20% O2 and 20% CO2)). All the flasks are placed overnight (about 16 hours) at 37° C. then 1 hour in modified atmosphere according to the preceding conditions at 37° C. One flask per condition is placed directly at +4° C. for 13 days and the other at 30° C. for 2 days, then at +4° C. for 11 days (13 days in total). At the end of this period, the cells were heate...

example 2

Mesenchymal Cells

Materials and Methods

[0087]In this example, and for the sake of clarity, only mesenchymal cells (MCs) capable of forming adherent fibroblast-like cell colonies (CFU-F) were considered.

[0088]Previously frozen mesenchymal cells produced from human bone marrow (cells retained on filters used for bone marrow filtration before transplantation) were thawed, cultured (α MEM, 10% FCS, β FGF (1 ng / mL), L-glutamine (2 mM), 0.5% penicillin-streptomycin) at 37° C. and 5% CO2 in 9 flasks and amplified to confluence. The flasks are then vented at 37° C. as follows: i) air (21% O2, 0.01% CO2); ii) hypoxia / hypercapnia (HH) (9% CO2, 5% O2); iii) hypoxia (5% O2, 0.001 CO2); iv) hypercapnia (20% O2 and 20% CO2) (2 flasks per condition; the 9th flask being used for analysis of viability / apoptosis and for the clonogenic test before preservation in hypothermia). For each condition, the first flask was placed directly at +4° C. and preserved at 4° C. for 5 days and the second was maintain...

example 3

Cells of the FDCPmix Cell Line

Materials and Methods

[0092]FDCPmix cells are amplified at 37° C. and 5% CO2 in RPMI medium supplemented with 20% horse serum and 8% WEHI-conditioned medium (source of interleukin-3). The cells are then divided into 2 flasks per condition under the following conditions: i) air (21% O2, 0.01% CO2); ii) hypoxia / hypercapnia (HH) (9% CO2, 5% O2); iii) hypoxia (5% O2, 0.001 CO2); iv) hypercapnia (20% O2 and 20% CO2). The protocol followed is the same as for mesenchymal cells in Example 2.

Results

[0093]Maintenance of FDCPmix cells is poor in air and in hypoxic / hypercapnic conditions without a period in moderate hypothermia (directly at +4° C.).

[0094]On the other hand, the combination hypoxia / hypercapnia or hypercapnia alone with a period in moderate hypothermia provides very good maintenance with about 60% of the FDCPmix cells (FIG. 5).

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Abstract

The present invention relates to a method for preserving, preferably human, cells, tissues or organs in severe hypothermia, comprising a step during which the cells, tissues or organs are kept in moderate hypothermia and preferably in a hypoxic and / or hypercapnic atmosphere, before placing them in severe hypothermia.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application is the U.S. national stage application of International Patent Application PCT / FR2016 / 050948, filed Apr. 22, 2016.[0002]The present invention relates to a novel method for medium and long-term preservation of cells, tissues or organs. This method is more particularly intended for the preservation of transplants until their transplantation in the recipient.BACKGROUND OF THE INVENTION[0003]To date, cryopreservation is the only method available for medium- and long-term preservation of hematopoietic cell transplants. Indeed, without freezing and at a temperature of 4° C., it was observed that the number and the functional capacity of progenitor cells contained in transplants decreased drastically after only 3 days of preservation (Hechler et al., 1996).[0004]Cryopreservation, however, has notable disadvantages, particularly potential agglutination during thawing or the risk of anaphylactic reaction triggered by the presence o...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): A01N1/02
CPCA01N1/0284A01N1/0242A01N1/0226A01N1/0252A01N1/0263A01N1/02
Inventor IVANOVIC, ZORANGERBY, SANDIEVLASKI-LAFARGE, MARIJA
Owner ESTAB FR DU SANG
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